1. Field of the Invention
The present invention relates to the fluorometric and electrochemical detection of low levels of primary amines and more particularly, to biogenic and endogenous amines such as catecholamines, amino acids, and peptides, by chemically converting the non-fluorescing amine of interest to an entity which is amenable to both fluorescent and electrochemical detection. Such is achieved by reacting fluorogenic reagents having an aromatic dialdehyde ring with the primary amine in the presence of a cyanide ion under mildly alkaline conditions. The invention further relates to the fluorometric and electrochemical detection of trace levels of cyanide.
2. Description of the Prior Art
Assaying techniques wherein a fluorogenic reagent is reacted with a substrate to form a fluorescing complex have been known for some time. One reagent used specifically for the derivatization of primary amines has been o-phthalaldehyde (OPA) which is of the formula: ##STR2## Under mildly alkaline conditions in the presence of a thiol, R.sub.1 SH and a primary amine R.sub.2 NH, OPA forms 1-alkylthio-2-alkylisoindole (AAI), which is a highly fluorescent adduct of the formula:
Although the fluorophores generated by the reaction of primary amines and OPA exhibit a relatively high fluorescent intensity with respect to many primary amines, it has been observed that the fluorescent intensity of the isoindole derivatives of primary amines containing an .alpha.-amido group are substantially lower. Thus, the OPA/thiol derivatizing system is not useful for the detection of femtomole quantities of peptides and proteins. Such represents a significant drawback of OPA for assaying many biological systems.
Another problem encountered with fluorogenic assaying techniques employing OPA relates to the relative instability of the 1,2-disubstituted isoindoles of certain amines such as glycine, .gamma.-amino butyric acid (GABA), and .beta.-alanine. These adducts have been observed to readily degrade into non-fluorescent products thereby placing severe time constraints on a practitioner when a concentration profile of the above amino acids is desired.
Naphthalene-2,3-dicarboxaldehyde (NDA) of the formula: ##STR3## has also been used as a fluorogenic reagent. However, its use has been limited to the detection of arginine and arginine methyl ester and, even in that limited case, it is only very recently that the structure of the fluorescent reaction product has been characterized.